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Effect of psoralen on the expression of PPARγ, osteocalcin, and trabecular bone area in rabbits with steroid-induced avascular necrosis of the femoral head

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R E S E A R C H A R T I C L E

Open Access

Effect of psoralen on the expression of

PPAR

γ

, osteocalcin, and trabecular bone

area in rabbits with steroid-induced

avascular necrosis of the femoral head

Huiying Li

*

, Dongfang Meng, Xiaorui Zhang and Dong Yuan

Abstract

Objective:Psoralen is a natural plant toxin which has the function of protecting fungi, insects, and herbivores. In this study, we aim to investigate the effect and mechanism of psoralen on steroid-induced avascular necrosis of femoral head (SANFH).

Methods:Thirty rabbits were randomly divided into blank group (n= 10), model group (n= 10), and experimental group (n= 10). Rabbits in blank and model groups were treated with normal saline, and rabbits in experimental group were treated with psoralen. Total RNA of bone marrow was extracted by trizol, and the mRNA expression of PPARγand osteocalcin were detected by q-PCR. Then, the mRNA expression of PPARγand osteocalcin in the three groups were compared. Western blot was used to detect the PPARγprotein expression in the bone of rabbits. ELISA was used to measure the osteocalcin protein.

Results:The mRNA expression of PPARγin model group significantly increased compared with blank group. The mRNA expression of osteocalcin in model group decreased compared with blank group. However, the mRNA and protein expressions of PPARγin experimental group were significantly decreased compared with the model group. The protein expressions of osteocalcin increased compared with the model group. There was no significant difference of trabecular bone area (TBA) between experimental and blank groups (P> 0.05). TBA in model group was lower than the experimental group (P< 0.05). There was no significant difference of TBA between experimental and blank groups (P> 0.05).

Conclusion:This research confirms that psoralen plays a positive role in the rehabilitation of SANFH.

Keywords:Steroid-induced avascular necrosis of the femoral head, Psoralen, Trabecular bone area, Bone marrow, Osteocalcin, PPARγ

Background

Avascular necrosis of the femoral head (ANFH) can occur in patients who receive hormonal therapy, causing steroid-induced avascular necrosis of the femoral head

(SANFH) [1]. SANFH is considered to be one of the

most common hip pain diseases. The disease can be di-vided into two categories: traumatic and non-traumatic. The former is mainly caused by hip trauma, such as femoral neck fracture and hip dislocation. The latter is

mainly caused by the use of corticosteroids and

alco-holism in China [2]. The causes of high-dose hormones

affecting blood viscosity are congenital dysplasia of hip vessels, sclerotin, and biologic anatomy. Increased blood viscosity may cause vasospasm of the femoral head, even lead to insufficient blood oxygen supply of the femoral head, and then lead to loss of osteocyte

ac-tivity and apoptosis [3]. The situation does not have

significant improvement in the short term, resulting in fracture and even microfracture of the trabecular bone in the weight-bearing area of the femoral head.

* Correspondence:huiyingli_hntcm@126.com

Orthopedics Department, The First Affiliated Hospital of Henan University of Chinese Medicine, 19 Renmin Road, Zhengzhou 450000, Henan, China

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ANFH that caused by the use of hormones is non-traumatic, and its pathogenesis is difficult to de-tect. The main symptom is pain of hip and groin, which is easy to be neglected and misdiagnosed; thus, it is easy to miss the best time of treatment. At present, all kinds of diseases are treated by hormone pulse therapy to cure patients and control their condi-tion. However, this treatment can lead to long-term ac-cumulation of hormones and significant increase in blood viscosity and vascular infarction in patients lead-ing to the bone formation decreased, the bone trabecu-lar turned sparse, the sclerotin dropped, the fracture fragments accumulated rarefaction of bone; and finally,

steroid-induced femoral head formed [4]. ANFH is

dif-ficult to find in its early stage, and due to the relatively poor medical conditions in some areas, some patients may be misdiagnosed and delayed to be cured. In addition, the disease course is long, and its late disabil-ity rate is high. Therefore, in order to further clarify the etiology, it is necessary to do early diagnosis and take the treatment as soon as possible, so as to allevi-ate the suffering of patients.

Psoralen is the dry and ripe fruit ofPsoralea

corylifo-lia L. Pharmacological studies have found that

psor-alen has anti-cancer activity which can enhance immunity and promote bone growth, and it also plays

the role of bacteriostasis and anti-psoriasis [5]. Some

research reported that psoralen promotes cartilaginous extracellular matrix (ECM) synthesis, as well as in-creased cartilaginous gene expression, and it may be a useful bioactive component for activating the

cartil-aginous cellular functions of chondrocytes [6]. Based

on the present condition, an in-depth study on SNAFH was to be made, with the advantages of traditional Chinese medicine combined with the advanced med-ical research methods. On the basis of the research, safe and effective drugs can be made to apply in clinic,

reduce the patients’ economic burden and pain, at the

same time reduce disability rate, and promote the de-velopment of traditional Chinese medicine as well as modern scientific theory.

Materials and methods Experimental animal

Approval for the experiment was obtained from the First Affiliated Hospital of Henan University of Chinese Medicine, (Approval number: YFYDW2016032), and all procedures were conducted in conformity with the guidelines and regulations of the First Affiliated Hos-pital of Henan University of Chinese Medicine. A total of 30 healthy adult New Zealand big ear white rabbits were selected in this research, male and female each 15,

weighing 2.5–3.0 kg for each rabbit. The rabbits were

purchased from Ji’nan Jinfeng Experimental Animal

Center (license No. SCXK (Lu) 20140006) and were fed by pellet feed, each rabbit a cage. Feed was purchased from Henan Animal Experiment Center (license No. SCXK (Yu) 2015-0005). Psoralen was purchased from Anhui Pu Ren Chinese Herbal Medicine Co., Ltd. (batch No.: 160627).

Methods

Grouping and intervention

The New Zealand white rabbits were fed in different cages and were freely to drink water with the normal diet. The humidity was about 50%. The room temperature was con-trolled between 23 °C and 25 °C. The ventilation was good, and drugs were administrated to the rabbits if there is no obvious abnormality after 1 week’s feeding.

The selected rabbits were randomly divided into three groups after being quarantined and observed for 1 week and were determined if there was no abnormality: blank

group (n= 10), model group (n= 10), and experimental

group (n= 10). After conducting the t test to rabbits’

weight and number of male and female in three groups, as well as confirming there was no significant difference, the

experiment was started (Fig. 1). All rabbits in the model

group were prevented from anaphylactic shock and rou-tinely given horse serum (Zhengzhou Yi Kang Bioengin-eering Co. Ltd) to establish osteonecrosis of the femoral head model. The rabbit model of alcohol-induced ONFH was successfully prepared by intravenous injection of horse serum. Compared with the pure alcohol gastric

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perfusion, the success rate of this method was higher, and the pathological changes of ANFH were more obvious. The lesions were concentrated in the subchondral area of femoral head [7]. The specific applications were as follows: the first application of the horse serum dose was 10 mL/ kg, through the ear vein injection into the blood, fed 3 weeks conventionally after the drug administration; the second time was also administered by the ear vein, and the horse serum was 6 mL/kg. Then, the rabbits were still fed regularly in the last 2 weeks, and the rabbits should be observed to see whether they have any changes. Two weeks later, we conducted hormone modeling with methylprednisolone (Hubei Fangleda Biochemical Co. Ltd) intraperitoneal injection, and sustained given medication for 3 days according to the dose of 45 mg/ kg, injected once a day. During the period of hormone administration, 100,000 units of penicillin were admin-istered to each rabbit every day for seven consecutive days to prevent infection. The rabbits of blank group were fed as usual. In the experimental group and blank

group, every rabbit was given (6 g L−1) 35 mg/kg

psor-alen (Anhui Pu Ren Chinese Herbal Medicine Co. Ltd) extract daily. Rabbits in each group were given intra-gastric administration for 8 weeks. They were weighed once a week and all of them were fed normally.

Materials

Eight weeks later, rabbits in each group completed treatment with drugs. Their skins were incised after intravenous anesthesia through the ear vein, and the bilateral femoral heads were exposed after the tissue separation. Visual observation of the femoral head shape was conducted to find out whether the bones were loose or not and whether the weight-bearing area of the femoral head was collapsed or not. Bilateral femoral heads were cut, and the femoral heads were incised from the middle of coronal plane. We rapidly aspirated the red bone marrow with a 5 ml syringe, and it was placed in a sterile centrifuge tube filled with DMEM culture medium.

Extraction of osteocalcin mRNA and PPARγ mRNA

was done as follows: the total RNA of bone marrow was extracted by trizol method, and the expression of

PPARγ mRNA and osteocalcin mRNA was detected by

fluorescence quantitative PCR instrument according to

the reagent box instruction. The expression of PPARγ

and osteocalcin protein was detected by western blot and ELISA.

The ratio of trabecular bone area (TBA) was obtained as follows: all HE-stained slices were observed × 40 in an optical microscope, in randomly selected five fields of vision, and photographed. Then, the Motic Image Advanced 3.2 analysis software was used to calculate

the TBA in the field of vision, calculate the fraction of TBA, and take the average of the TBA ratio.

Statistical analysis

All the data were analyzed and processed by SPSS19.0 statistical analysis software. The measurement data

were expressed by (x ± s), and one-way ANOVA was

used to analyze comparison between groups. The LSD method was used to compare the variances between groups. Once the variance was not uniform, we then

immediately replaced LSD method with Dennett’s T3

method. There was a significant difference atP< 0.05.

Results

Psoralen improved the pathological changes of steroid-induced osteonecrosis of the femoral head

The morphological changes of adipocytes were ob-served as follows: when primary cells were inoculated for about 24 h, the cells began to adhere to the wall, showing a circular undifferentiated state. After 3 to 4 days, the cells became spindle deformed and the vol-ume gradually increased, forming fibrous cell morph-ology and began to split and proliferate. After 7 days, the volume of cells increased, which was fusiform, tri-angular, or irregular. After 10 days, the proliferation of cells was significantly accelerated, with most of them clustered along a certain direction and with a few ir-regular arrangements. After 14 to 16 days, the cells

ag-gregated into layers and showed colony growth (Fig. 2a

blank group, b model group, c experimental group). The average diameter of adipocytes in the experimen-tal group and blank group was at a low level (38.00 ± 2.94 and 29.50 ± 3.10), without significant difference

be-tween the two groups (P> 0.05). The average diameter

of adipocytes in the model group was higher (52.25 ± 4.78), with a significant difference compared with the experimental group and the blank group (P< 0.05).

The pathological change of HE staining is shown in

Fig. 3 (a blank group, b model group, c experimental

group). Psoralen significantly improved the structure of avascular necrosis of the femoral head in experimental group.

Psoralen decreased PPARγand increased osteocalcin expressions in steroid-induced osteonecrosis of the femoral head

The relative expression of PPARγ mRNA and

osteocal-cin mRNA was analyzed by comparison of the 2−△△Ct

[5]. GAPDH was used as an internal gene to calculate

the △Ct values of each group (the Cttarget gene

sub-tracted the Ctinternal gene), and the △△Ct value was

ob-tained by comparison between groups (the two groups

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objective genes between groups were analyzed by the value of 2−△△Ct: 2−△△Ct ≥2, the expression of compara-tive target gene between the groups was significantly

increased; and 2−△△Ct≤0.5, the expression of

compara-tive target gene between the groups was significantly decreased. The results showed that compared with the

blank group, the relative expression of PPARγ mRNA

in the model group was significantly increased, and the expression of osteocalcin mRNA was significantly

de-creased. Compared with the model group, the PPARγ

mRNA of the experimental group was significantly de-creased and the expression of osteocalcin mRNA was significantly increased (Table1).

From Table 2, TBA, the single factor variance test

showed that there was a significant difference between

the groups in TBA (P< 0.05). Compared with blank

group and experimental group, the TBA of model group was at the lowest level with a significant

differ-ence (P< 0.05). TBA of experimental and blank groups

were high with no significant difference between groups (P> 0.05).

The result of western blot showed that PPARγ

pro-tein was significantly decreased by psoralen (Fig.4a, b). The ELISA result also showed that psoralen increased

osteocalcin protein in experimental group (Fig.4c).

Discussion

At present, the etiology of ANFH is relatively clear, which is due to prolonged and/or high-dose use of

glucocorticoid-induced osteonecrosis of the femoral head. SANFH is a metabolic disease that occurs due to the use of glucocorticoid drugs, leading to impaired blood supply to the femoral head and death of bone cells and bone marrow composition, which in turn lead to structural change, collapse of the femoral head, and

articular dysfunction [8]. But in its pathogenesis, the

medical profession has not yet had clear conclusions and the following several theories have been widely recognized.

Theory of lipid metabolic disorders

Prolonged high-dose use of hormones can increase blood lipids and cause hyperlipidemia, leading blood tends to be stagnated. The fat embolism situation of the whole body is serious, and the femoral head tiny ar-teries form adipose suppository, which directly causes

the partial blood stasis [9]. The bone marrow cells are

occupied by adipose cells, and the adipose cells fuse into pieces, inducing the death of marrow-derived cells, resulting in ANFH.

Theory of intraosseous pressure increasing

As early as 1981, Bünger et al. suggested that the in-crease of intraosseous pressure is the pathogenesis of

ANFH [10]. The medical profession believed that the

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vessels spasm caused ischemia, and then form ANFH. But at the same time, some scholars pointed out that the increase in intraosseous pressure in the middle and late stage of the disease was a secondary change, and early ANFH did not appear in the intraosseous pressure phenomenon. Although there is no obvious evidence that indicates that the increase in intraosseous pressure is the primary or secondary change of ANFH, the the-ory of intraosseous hypertension has been universally recognized.

Theory of osteocyte apoptosis

The medical profession generally believes that the use of glucocorticoid in excess of conventional doses can cause ANFH. Studies have shown that high-dose applications

of glucocorticoids can decrease the activity of osteoblasts and increase the apoptosis of mature bone cells. The number of osteoblasts decreased significantly, and the trabecular bone was sparse, which resulted in osteopor-osis [11]. Eerhardt et al. suggested that the staining sec-tion of rabbits with SANFH model could be observed under the light microscope, and the trabecular bone of the subchondral bone in the femoral head became thin-ner. Apoptosis of bone cells was prevalent in special staining around the trabecular bone [12].

Osteoporosis theory

The negative effects of long-term use of glucocorticoids include osteoporosis. Glucocorticoids can overdecom-pose proteins and reduce their synthesis, resulting in

Table 1Expression of PPARγmRNA and osteocalcin mRNA in each group (x ± s)

Group GAPD

(Ct value)

PPARγ Osteocalcin

Ct △Ct 2−△△Ct Ct △Ct 2−△△Ct

Blank group 24.00 ± 2.32 33.54 ± 0.99 9.54 ± 1.50 28.37 ± 1.96 4.37 ± 2.03

Model group 27.06 ± 2.13 34.28 ± 0.46 7.21 ± 1.77 5.02 32.51 ± 1.97 5.44 ± 1.07 0.47

Experimental group 25.47 ± 2.65 33.95 ± 1.69 8.48 ± 1.55 0.42 29.70 ± 2.37 4.23 ± 1.37 2.32

Note: There are significant differences in the same column data for different characters

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the thinning of the trabeculae bone, at the same time against vitamin D, and reducing calcium absorption in the gastrointestinal tract, thus causing large amounts of cal-cium to be excreted. Taking large doses of steroid-like drugs are prone to become bone hyperplasia, which can reduce the number of trabecular bone and osteoblasts, thus lead to osteoporosis, and ultimately lead to the col-lapse of the femoral head bearing area, and even necrosis [13]. In addition, in the process of differentiation of bone marrow stromal cells into adipocytes, hormones play a certain role in inducing the reduction of differentiation of osteoblasts. These are the traditional Chinese medicine factors that lead to osteoporosis and ANFH.

At present, the treatment methods of SANFH are di-versified and the indications are various. And the treat-ment of this disease is mainly divided into non-surgical

therapy and surgical therapy, and surgical therapy can be broadly divided into two categories: minimally inva-sive and joint replacement. The medical profession adopts treatment options mainly based on different in-dications and patient wishes and their physical and eco-nomic conditions. But it is aimed to improve the quality of life of patients. The hip joint is the main weight-bearing joint in the human body, and to reduce or avoid the hip joint weight is the main concern in the course of treatment, so as to obtain the maximum clin-ical effect. The surgclin-ical treatment has made a great pro-gress with the increasing incidence of SANFH and development of modern medical research. Depending on the progression of the course, different methods of operation can be chosen. At present, the commonly used surgical methods in clinical practice are as follows: (1) the ARCO classification determines that the femoral head has not yet collapsed and can be conducted fem-oral head drilling decompression plus bone graft or femoral head drilling decompression plus tantalum rod support surgery. The purpose is to delay the collapse of the weight-bearing area of the femoral head, even with-out collapse. The operation is simple and widely used in young and middle-aged people. (2) Bone flap trans-plantation surgery is used relatively less in recent years; it is mainly used to promote bone flap and blood vessel

Fig. 4Results of western blot and ELISA.a,bThe result of western blot showed that PPARγprotein was significantly decreased by psoralen.cThe ELISA result also showed that psoralen increased osteocalcin protein in experimental group

Table 2Effect of psoralen on trabecular bone area in each group (x ± s) (n= 10)

Group Number Ratio of trabecular

bone area

Blank group 10 45.23 ± 3.53

Model group 10 34.72 ± 2.98

Experimental group 10 41.45 ± 3.18

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crawling and the growth of new bone and to delay the time of joint replacement. (3) One of the new surgical methods in recent years, stem cell transplantation sur-gery, is also popular, but because of its high technical requirements and high cost, results in the range of use is very small, but the scope of surgery and the oper-ation can promote the repair of ANFH, so the experi-mental research is also increasing. (4) Joint replacement is currently the most widely used surgical method in middle-aged and old people; the surgical technique is mature, the cost is relatively low, and the quality of life improved obviously. For the use of traditional Chinese medicine, every theory has its own unique insights. But generally speaking, tonifying liver and kidney and strengthening bones and muscles have been generally

recognized [14]. In addition, long-term application of

hormones leads to hyperlipidemia, a systemic fat em-bolism. And small artery lumen at the end of the

fem-oral head cartilage is rare [15]. The fat clumps that

adhere to the inner wall of the vessel form fat embol-ism. Bone marrow cells are occupied by fat cells. Fat cells fuse into pieces, resulting in hemopoietic cells in the bone marrow dead. Based on this theory, many fac-tors, such as lipid-lowering drugs, anticoagulant blood, and vascular relaxant, which are used to control the ne-crosis of the femoral head caused by the proliferation of adipocytes, are widely used in clinical treatment [16]. However, there is no clear and unified view on the usage, dose, and the application time.

PPARγ mRNA mainly plays a role in regulating fat, so

it has great significance in the process of adipogenesis of cells in blood vessel and bone marrow [17,18]. Excessive differentiation of the adipose tissue leads to sparse tra-becular bone, where bone fat cells are multiplied, result-ing in osteoporosis, which can lead to minor fractures of

bone in severe cases [19, 20]. Through the experimental

study on the adipogenic effect of bone marrow stromal

stem cells from osteoporosis rats, Liu et al. [21] found

that regulating and inhibiting the expression of PPARγ

mRNA can effectively reduce the incidence of osteopor-osis. At the same time, it also confirmed that the low

ex-pression of PPARγmRNA could reduce the risk of small

fracture and reduce ANFH.

Osteocalcin mRNA is one of the most sensitive

in-dexes in the process of osteogenic differentiation [22].

The increase of its activity can accelerate the process of ALP activity combined with it and thus promote the

bone formation of osteoblasts [23]. On the other side,

the combination of osteocalcin and calcium ions has a positive effect in promoting bone deposition and bone

growth. Peng et al.’s exploration from the side reflects

the activity of osteocalcin having a positive correlation with osteoblast differentiation and osteoclast apoptosis

[24]. Zhu et al. believe that one of the bone secretion

hormones is osteocalcin, which regulates the pathogen-esis of osteoblasts and osteoclasts and regulates

adipo-cytes by converting osteoclasts into blood [25].

Therefore, the high expression of osteocalcin can effect-ively reduce the differentiation of adipocytes, increase bone matrix, and strengthen sclerotin [26,27].

Conclusions

In clinic, because of the treatment of diseases, many pa-tients have to take a lot of hormones for a long time, which leads to SANFH. After discovering the effect of

Psoraleaon the treatment of ANFH through animal ex-periments, we applied the dialectical thinking of trad-itional Chinese medicine and took this drug as the main drug (sovereign drug) combined with other trad-itional Chinese medicine to constitute the tradtrad-itional Chinese medicine prescription for the prevention and treatment of SANFH. The medicine in the prescription was decocted and boiled together and taken by the pa-tient for different times depending on the papa-tient’s con-dition. The results were satisfactory, especially for patients with early SANFH.

In this experiment, compared with blank group, the

relative expression of PPARγ in the model group was

significantly increased, and the expression of osteocal-cin was significantly reduced; compared with model

group, the PPARγ of the experimental group was

sig-nificantly decreased and the expression of osteocalcin was significantly increased. This experiment confirms that psoralen can reduce the adipogenesis of cells in bone marrow, promote the deposition of calcium, pvent osteoporosis, and play a positive role in the re-habilitation of ANFH.

Abbreviations

ANFH:Avascular necrosis of the femoral head; SANFH: Steroid-induced avascular necrosis of femoral head; TBA: Trabecula bone area

Acknowledgements

Not applicable.

Funding

This research was supported by Key research projects of higher education in Henan province (grant number: 15A360004).

Availability of data and materials

All data generated or analyzed during this study are included in this published article.

Authors’contributions

HL and DM designed the study and prepared the first draft of the paper. HL is guarantor. XZ and DY contributed to the acquisition of the data. DM, XZ, and DY were responsible for statistical analysis of the data. DM and DY were responsible for the interpretation of data. All authors revised the paper critically for intellectual content and approved the final version. All authors agree to be accountable for the work and to ensure that any questions relating to the accuracy and integrity of the paper are investigated and properly resolved.

Ethics approval and consent to participate

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Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Received: 31 October 2018 Accepted: 27 December 2018

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Figure

Fig. 1 Grouping
Fig. 2 Adipocytes. a Oil red O staining × 100 of experimental group. b Oil red O staining × 100 of blank group
Table 1 Expression of PPARγ mRNA and osteocalcin mRNA in each group (�x ± s)
Table 2 Effect of psoralen on trabecular bone area in eachgroup (�x ± s) (n = 10)

References

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